Receptor-tyrosine kinase inhibitors (TKI), sorafenib (SRF) and sunitinib (SNT) are clinically used in treatment of RCC. Both drugs have anti-proliferative effects in several tumor types with inhibition of angiogenesis being a major mechanism of action in RCC. The intracellular molecular effects of SRF and SNT have not been completely described. Thus the identification of targets mediating the anti-proliferative effects of SRF and SNT could be useful to improve efficacy and to reduce toxicity. In this work we used immunoaffinity isolation for enrichment of phosphotyrosine containing proteins in control or SRF-treated CAKI-1 cells. To identify tyrosine-phosphorylated proteins that were differentially affected by SRF treatment, we performed liquid chromatography - tandem mass spectrometry (MS) followed by quantitative analysis using peptide counts and native peptide reference MS methods. In CAKI-1 cells, MS results revealed that signal transducer and activator of transcription 1 (STAT1) and Ras and Rab interactor 1 (RIN1) were the predominant proteins which exhibited tyrosine hypophosphorylation following treatment for 48 h with 10 μM SRF, when compared to the untreated control. Western blot analysis with phosphospecific antibody indicated a potential role for tyrosine 36 phosphorylation in RIN1 (pY36-RIN1) in CAKI-1, RC-45 and 786-O clear cell RCC. Up to a 8-fold SRF dose-dependent (5-15 µM) decrease in pY36-RIN1, but not in total RIN1 expression was also observed in a panel of RCC cell lines. Hypophosphorylation of Y36-RIN1 was also detected in CAKI-1 cells treated with 5 µM SNT but not with imatinib (≤ 10 µM), suggesting that pY36-RIN1 is a selective target for agents SRF and SNT that are also clinically effective in treatment of RCC. A ~2-fold higher expression of RIN1 (total and phosphorylated) was observed in CAKI-1 cells following continuous exposure (~ 6 weeks) to 7.5 µM SRF. However, unlike parent CAKI-1 cells, in cells adapted to 7.5 μM SRF, exposure to 10 μM SRF did not completely abrogate phosphorylation of Y36-RIN1. Stable expression of RIN1 targeted shRNA in CAKI-1 cells led to a 2-4 fold down-regulation of RIN1 protein and significantly (p < 0.002) enhanced sensitivity to the anti-proliferative effects of 7.5 μM SRF or SNT compared to scrambled shRNA. In contrast, overexpression of RIN1 in RC-45 or 786-O RCC cells resulted in significantly (p < 0.05) increased resistance to 10 μM SRF or 7.5 μM SNT compared to vector control. These results demonstrate that pY36-RIN1 is targeted by SRF and SNT and strongly suggest that anti-proliferative effects of these TKI drugs in clear cell RCC is mediated by inhibitory effects on RIN1 tyrosine phosphorylation.